One of the primary toxic metabolites of alcohol (ethanol) metabolism, acetaldehyde causes protein adducts and cellular dysfunction at the molecular level, and acetaldehyde accumulation causes facial flushing, tachycardia, headache and nausea, and alcohol avoidance at the physiological level. Acetaldehyde is primarily cleared by aldehyde dehydrogenases, namely ALDH2, ALDH1B1, and ALDH1A1. ALDH2 has been well characterized and a well-known inactivating polymorphism, ALDH2*2 has been found to affect approximately 50% of some Asian populations. ALDH1B1 has been partially characterized more recently, but there is much to learn. Recent epidemiological studies have identified polymorphisms of ALDH1B1 which are associated with alcohol avoidance and alcohol hypersensitivity reactions;this is the first such variant in acetaldehyde clearance reported in Caucasian populations. In addition to the alcohol metabolism pathway, we also hypothesize that ALDH1B1 may play a role in the bioactivation of nitroglycerin and retinoic acid;the latter could have significant implications in cancer biology. Given these roles, we propose a comprehensive characterization of ALDH1B1 using a broad range of techniques including molecular modeling, enzyme kinetics, metabolomics, high throughput screening of inhibitors and activators, and site directed mutagenesis to better understand multiple dynamic aspects of this potentially critical enzyme. First a more complete spectrum of substrates for ALDH1B1 will be tested including those which we hypothesize from previous evidence (nitroglycerin and retinaldehyde), and those which are determined by unbiased metabolomic approaches. To better allow manipulation of ALDH1B1 levels in experimental systems, an inhibitor and activator profile will be determined, first using known ALDH inhibitors, and later using high throughput screening techniques. It is likely that ALDH1B1 is phosphorylated, and we will use spectrometry combined with kinases and phosphatases to determine if ALDH1B1 is phosphorylated, and if so, whether these modifications alter enzyme activity levels. Finally, the three variants of ALDH1B1 which are nonsynonymous and present at a frequency of at least 1% in human populations will be created through site-directed mutagenesis, expressed in a baculovirus system, and characterized for enzyme activity to better understand what effects these potentially inactive enzymes will have in individuals who carry them. These experiments will help to better understand the effects that differential acetaldehyde metabolism may have on Caucasian populations, as well as clarifying the other, diverse roles of ALDH1B1. PUBLIC HEALTH RELEVANCE: In this study we propose to characterize the effects of an enzyme (ALDH1B1) which modulates the extent of alcohol symptoms and toxicity. Similar to ALDH2*2, a variant in Asian populations which causes facial flushing and increased nausea and headaches after consuming alcohol, ALDH1B1 has polymorphisms which may affect the alcohol sensitivity of Caucasian populations. Better characterization of this critical enzyme will improve public health by allowing a more complete understanding of alcohol sensitivity and susceptibility to alcoholism.